PROJECT SUMMARY/ABSTRACT There is a great deal of interest in adipose biology, particularly in light of the world-wide epidemic in obesity and metabolic diseases, including type 2 diabetes, cardiovascular disease and cancer. While adipose tissues are best known as the major storage site for calories, certain fat tissues play a critical role in adaptive thermogenesis, the process whereby chemical energy is dissipated in the form of heat in response to external stimuli. Thermogenic adipose tissues, brown and beige, defend the body against hypothermia, obesity and other metabolic disorders. Critical unmet needs include understanding the detailed molecular pathways by which chemical energy is converted into heat and the discovery of human therapeutics that might increase amounts and function of thermogenic fat. Four years ago, we described a previously unknown thermogenic pathway in brown and beige fat that plays a major role in both energy expenditure and suppression of obesity in animal models. Disruptions of this futile creatine cycle causes levels of obesity not observed with ablations of any previously described thermogenic mechanisms, including UCP1; in response to these observations, I am focusing this grant entirely on further biochemical and physiological studies of this futile creatine pathway. One Aim will focus on the role of the creatine transporter (CrT) in fat tissues, where preliminary data with adipo-CrT KO mice shows that this exogenous pathway for creatine accumulation contributes significantly to whole body energy homeostasis. The physiological role of the CrT specifically in fat will be analyzed with metabolic cages to study mutant mice under several different physiological perturbations. This mutation will also be combined with our previous genetic model (adipo-GATM-KO), which is unable to synthesize creatine de novo, to create an animal model totally lacking adipose accumulation of creatine. A related Aim will be to study regulation of the CrT mRNA and protein; preliminary data shows mRNA to be down-regulated in fat cells from obese human subjects. Importantly, we will also use metabolomic studies (LC/MS) to follow the fate of phosphocreatine (CrP), as it is processed/hydrolyzed in mitochondria from thermogenic fat cells. Our last Aim will focus on a major unanswered biochemical question: exactly how is the high energy phosphate on CrP dissipated as part of this futile cycle. In this regard, we have exciting preliminary data using 31P NMR: mitochondrial preparations from thermogenic fat contain an activity that can hydrolyze CrP directly. We have purified this activity and have identified it as TNAP, an alkaline phosphatase. While not annotated as a mitochondrial protein, we find a substantial portion of this protein in the mitochondrial associated membrane (MAM) fraction. We will perform genetic and pharmacological manipulations of TNAP to determine its role in thermogenesis and the futile creatine cycle. We will also use protein Mass Spectrometry to determine how this protein may be modified to achieve its association with mitochondria. Together, these studies will advance basic knowledge of adaptive thermogenesis and provide potential new avenues to human therapeutics in metabolic diseases.